本文選題：老年抑郁　切入點(diǎn)：血管新生　出處：《昆明醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：[目的] 建立慢性應(yīng)激老年大鼠抑郁模型,評(píng)估老年抑郁大鼠海馬血管新生的變化及其相關(guān)的分子機(jī)制,研究抗抑郁藥物文拉法辛的干預(yù)作用。 [方法] 1.SPF級(jí)雄性老年SD大鼠(20月齡)共72只。根據(jù)隨機(jī)數(shù)字表將大鼠隨機(jī)分為模型組、文拉法辛組、生理鹽水組、對(duì)照組各18只。模型組、文拉法辛組、生理鹽水組均給予經(jīng)改良的慢性不可預(yù)測(cè)的溫和應(yīng)激結(jié)合孤養(yǎng)共5w,文拉法辛組于應(yīng)激后第3w末開(kāi)始同時(shí)給予文拉法辛(5mg.kg-1.d-1)腹腔注射持續(xù)14d；生理鹽水組于應(yīng)激后第3w末開(kāi)始同時(shí)給予生理鹽水腹腔注射持續(xù)14d；對(duì)照組不給予任何處理,3只／籠正常喂養(yǎng)共5w。采用敞箱實(shí)驗(yàn)和蔗糖消耗實(shí)驗(yàn)評(píng)估大鼠抑郁模型建立情況,并觀察老年抑郁大鼠及文拉法辛治療后的行為學(xué)特征。 2.對(duì)上述大鼠標(biāo)本,采用HE染色觀察各組大鼠海馬的形態(tài)學(xué)變化。應(yīng)用免疫組織化學(xué)技術(shù)測(cè)定各組大鼠海馬的微血管密度(microvessels density, MVD)以評(píng)估老年抑郁大鼠及文拉法辛干預(yù)后的海馬血管新生的變化。 3.采用免疫組織化學(xué)技術(shù)測(cè)定各組大鼠海馬血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)、血管生成素1(angiopoietin1, ANG1)、堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor,bFGF)的蛋白表達(dá)變化。采用免疫印跡(western blot)和逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcript polymerase chain reaction, RT-PCR)技術(shù)測(cè)定各組大鼠海馬VEGF及其受體血管內(nèi)皮生長(zhǎng)因子受體2(vascular endothelial growth factor receptor2, VEGFR2)、ANG1及其受體內(nèi)皮細(xì)胞特異性酪氨酸受體2(tyrosine kinase with immunoglobulin and epide growth factor homology domain2, TIE2)、bFGF及其受體成纖維細(xì)胞生長(zhǎng)因子受體1(fibroblast growth factor receptor1, FGFR1)的蛋白及mRNA表達(dá)水平。以評(píng)估老年抑郁大鼠及文拉法辛干預(yù)后的海馬促血管新生因子表達(dá)的變化。 4.采用Western Blot和RT-PCR測(cè)定各組大鼠海馬的磷脂酰肌醇-3-激酶(phosphoinositide3-kinase, PI3K)、蛋白激酶B (protein kinase B, Akt)、Janus蛋白酪氨酸激酶1(janus protein tyrosine kinase1, JAK1)、信號(hào)轉(zhuǎn)錄與轉(zhuǎn)導(dǎo)活化因子3(signal transducer and activator of transcription3, STAT3)的蛋白及mRNA表達(dá)水平。并采用Western Blot測(cè)定磷酸化-Akt (phospho-Akt, p-Akt)蛋白表達(dá)水平。以評(píng)估老年抑郁大鼠及文拉法辛干預(yù)后的與海馬血管新生作用相關(guān)的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)分子的變化。 5.采用多元線性回歸分析,評(píng)估MVD與抑郁行為學(xué)指標(biāo)敞箱實(shí)驗(yàn)的水平運(yùn)動(dòng)得分、垂直運(yùn)動(dòng)得分、清潔時(shí)間和蔗糖消耗實(shí)驗(yàn)的蔗糖消耗量之間的關(guān)系。采用Bivariate相關(guān)分析,評(píng)估VEGF蛋白、ANG1蛋白、bFGF蛋白相互之間的關(guān)系；并評(píng)估MVD與VEGF蛋白、MVD與ANG1蛋白、MVD與bFGF蛋白之間的關(guān)系。采用一元線性回歸分析,評(píng)估MVD與VEGF蛋白、MVD與bFGF蛋白之間的關(guān)系。 [結(jié)果] 1.敞箱實(shí)驗(yàn)和蔗糖消耗實(shí)驗(yàn)顯示模型組、文拉法辛組和生理鹽水組抑郁模型均建立成功。敞箱實(shí)驗(yàn)結(jié)果：組內(nèi)比較：模型組、文拉法辛組、生理鹽水組于應(yīng)激后3w、應(yīng)激后5w分別與同組應(yīng)激前1d比較,模型組和生理鹽水組應(yīng)激后5w與同組應(yīng)激后3w比較,水平運(yùn)動(dòng)得分、垂直運(yùn)動(dòng)得分均降低(P均0.05),清潔時(shí)間縮短(P0.05)。組間比較：應(yīng)激后5w,與對(duì)照組比較,文拉法辛組的水平運(yùn)動(dòng)得分、垂直運(yùn)動(dòng)得分降低(P0.05),清潔時(shí)間縮短(P0.05)；模型組和生理鹽水組的水平運(yùn)動(dòng)得分、垂直運(yùn)動(dòng)得分明顯降低(P0.01),清潔時(shí)間顯著縮短(P0.01)；與生理鹽水組比較,文拉法辛組垂直運(yùn)動(dòng)得分增加(P0.05)。蔗糖消耗實(shí)驗(yàn)結(jié)果：組內(nèi)比較：模型組和生理鹽水組應(yīng)激后5w分別與同組應(yīng)激前1d、同組應(yīng)激后3w比較,蔗糖消耗、蔗糖偏愛(ài)減少(P均0.05)。組間比較：應(yīng)激后5w,與對(duì)照組比較,模型組和生理鹽水組的蔗糖消耗顯著減少(P0.01)；與生理鹽水組比較,文拉法辛組的蔗糖消耗增加(P0.05)。 2.HE染色顯示：對(duì)照組海馬錐體細(xì)胞層厚,排列整齊、緊密,毛細(xì)血管數(shù)量正常；模型組和生理鹽水組海馬錐體細(xì)胞層變薄,細(xì)胞間隙增大,排列紊亂、疏松,毛細(xì)血管數(shù)量減少；文拉法辛組海馬錐體細(xì)胞層薄,但細(xì)胞排列整齊、緊密,毛細(xì)血管數(shù)量明顯增多,管腔不規(guī)則。免疫組織化學(xué)染色結(jié)果顯示：與對(duì)照組比較,模型組和生理鹽水組海馬微血管密度降低(P0.05)；與生理鹽水組比較,文拉法辛組海馬微血管密度增加(P0.05)。 3.與對(duì)照組比較,模型組和生理鹽水組海馬VEGF蛋白及VEGFR2蛋白、VEGFmRNA及VEGFR2mRNA表達(dá)降低(P0.05)：與生理鹽水組比較,文拉法辛組海馬VEGF蛋白及VEGFR2蛋白、VEGFmRNA及VEGFR2mRNA表達(dá)增加(P0.05)。 4.與對(duì)照組比較,模型組和生理鹽水組海馬ANG1蛋白及TIE2蛋白、ANG1mRNA及TIE2mRNA表達(dá)增加(P0.05)；與生理鹽水組比較,文拉法辛組海馬ANG1蛋白及TIE2蛋白、ANG1mRNA及TIE2mRNA表達(dá)增加(P0.05)。 5.與對(duì)照組比較,模型組和生理鹽水組海馬bFGF蛋白及FGFR1蛋白、bFGFmRNA及FGFR1mRNA表達(dá)降低(P0.05)；與生理鹽水組比較,文拉法辛組海馬bFGF蛋白及FGFR1蛋白、bFGFmRNA及FGFR1mRNA表達(dá)明顯增加(P0.01)。 6.與對(duì)照組比較,模型組和生理鹽水組海馬P13K蛋白及Akt蛋白、p-Akt蛋白、PI3KmRNA及AktmRNA表達(dá)均下降(P0.05)；與生理鹽水組比較,文拉法辛組海馬P13K蛋白及Akt蛋白、p-Akt蛋白、PI3KmRNA及AktmRNA表達(dá)明顯增加(P0.01)。 7.四組海馬JAK1蛋白及STAT3蛋白、JAK1mRNA及STAT3mRNA表達(dá)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 8.在MVD與行為學(xué)結(jié)果的回歸分析中,敞箱實(shí)驗(yàn)的垂直運(yùn)動(dòng)得分系數(shù)有統(tǒng)計(jì)學(xué)意義(P0.05),回歸方程為MVD=5.056+0.580垂直運(yùn)動(dòng)得分。在VEGF蛋白、ANG1蛋白、bFGF蛋白的相關(guān)分析中,VEGF蛋白與ANG1蛋白、VEGF蛋白與bFGF蛋白均顯著相關(guān)(r分別為0.573、0.742,P分別為0.003、0.000)。在MVD與VEGF蛋白的回歸分析中,VEGF蛋白系數(shù)有顯著統(tǒng)計(jì)學(xué)意義(P0.01),回歸方程為MVD=3.391+11.507VEGF。在MVD與bFGF蛋白的回歸分析中,bFGF蛋白系數(shù)有顯著統(tǒng)計(jì)學(xué)意義(P0.01),回歸方程MVD=3.047+10.884bFGF。 [結(jié)論] 1.本實(shí)驗(yàn)成功建立了老年大鼠抑郁模型。老年抑郁大鼠有其抑郁樣癥狀特征：精神運(yùn)動(dòng)性遲滯癥狀突出,并隨應(yīng)激時(shí)間延長(zhǎng)而加重；興趣下降癥狀較晚才出現(xiàn)。相同劑量的文拉法辛針對(duì)不同癥狀療效存有差異：可遏制活動(dòng)遲滯癥狀的加重,但不能恢復(fù)到正常水平；可防治興趣下降癥狀出現(xiàn)。 2.慢性應(yīng)激老年抑郁大鼠的海馬微血管密度減少,血管新生可能受抑。文拉法辛干預(yù)后海馬微血管密度增加,對(duì)海馬血管新生可能有促進(jìn)作用。老年抑郁大鼠的海馬微血管密度與老年大鼠的抑郁樣癥狀有關(guān),提示老年抑郁障礙可能與血管損傷有關(guān)。 3.經(jīng)過(guò)持續(xù)5周的經(jīng)改良的慢性不可預(yù)測(cè)的溫和應(yīng)激結(jié)合孤養(yǎng),老年抑郁大鼠海馬VEGF及受體VEGFR2、ANG1及受體TIE2、bFGF及受體FGFR1的蛋白及mRNA表達(dá)可發(fā)生改變。經(jīng)文拉法辛14天抗抑郁治療可促進(jìn)老年抑郁大鼠海馬VEGF及受體VEGFR2、ANG1及受體TIE2、bFGF及受體FGFR1的蛋白及mRNA的表達(dá)。海馬VEGF蛋白與ANG1蛋白、VEGF蛋白與bFGF蛋白有關(guān)。海馬微血管密度與VEGF蛋白有關(guān),海馬微血管密度與bFGF蛋白有關(guān)。提示VEGF、ANG1、bFGF在血管新生的分子機(jī)制中可能參與了老年抑郁障礙的發(fā)病和文拉法辛的抗抑郁作用。 4.在與血管新生作用相關(guān)的細(xì)胞內(nèi)信號(hào)通路中,慢性應(yīng)激老年抑郁大鼠的海馬PI3K/Akt信號(hào)通路可能受到影響,文拉法辛可促進(jìn)PI3K/Akt信號(hào)通路的活化。JAK1/STAT3信號(hào)通路未發(fā)生明顯變化。提示PI3K/Akt信號(hào)通路在血管新生的分子機(jī)制中可能參與了老年抑郁障礙的發(fā)病及文拉法辛的抗抑郁作用。
[Abstract]:[Objective]
Objective to establish a chronic stress depression model in aged rats, evaluate the changes of hippocampus angiogenesis and related molecular mechanisms in elderly depression rats, and study the intervention effect of antidepressant venlafaxine.
[method]
1.SPF male aged SD rats (20 month old) and 72 in total. According to the random number table, the rats were randomly divided into model group, Vin Rafa Sin group, saline group and control group with 18 rats in each group. Model group, Vin Rafa Sin group, saline group were treated by modified chronic unpredictable Wen Heying induced by isolated feed 5W, Vin Rafa Sin group to stress after 3W starts at the end and give Vin Rafa Sin (5mg.kg-1.d-1) intraperitoneal injection duration 14d; saline group on stress after 3W began at the end of saline intraperitoneal injection for 14d; the control group was not given any treatment, 3 per cage normal feeding 5w. by open field test and sucrose consumption experimental evaluation of depressed rats, and observe the elderly depression rats and Vin Rafa Sin after treatment of behavioral characteristics.
2. of the big mouse, using HE staining to observe the morphological changes of hippocampus of rats in each group. The determination of microvessel density in hippocampus of rats by immunohistochemical technique (microvessels density MVD) to assess changes in rats and the geriatric depression venlafaxine treatment and hippocampi angiogenesis prognosis.
3. immunohistochemistry detected in vascular endothelial growth factor of rats (vascular endothelial, growth factor, VEGF), angiopoietin 1 (angiopoietin1, ANG1), basic fibroblast growth factor (basic fibroblast, growth factor, bFGF). The expression changes of protein by immunoblotting (Western blot) and reverse transcriptase polymerase chain (reverse transcript polymerase chain reaction reaction, RT-PCR) technique were used to detect the rat hippocampal VEGF receptor and vascular endothelial growth factor receptor 2 (vascular endothelial growth factor receptor2, VEGFR2, ANG1) and its receptors in endothelial cell specific tyrosine kinase receptor 2 (tyrosine kinase with immunoglobulin and Epide growth factor homology domain2, TIE2, bFGF) and its receptor into fibroblast growth factor receptor 1 (fibroblast growth factor receptor1, FGFR1) and mRNA protein To evaluate the expression of angiogenesis factor in the hippocampus of aged depressive rats and venlafaxine.
4. using Western Blot and RT-PCR were measured in rat hippocampus were phosphatidylinositol -3- kinase (Phosphoinositide3-Kinase, PI3K), protein kinase B (protein kinase B, Akt), Janus protein tyrosine kinase 1 (Janus protein tyrosine kinase1, JAK1), signal transduction and activation of transcription factor 3 (signal transducer and activator of Transcription3, STAT3 the expression levels of protein and mRNA). And the determination of phosphorylation of -Akt by Western Blot (phospho-Akt, p-Akt). The protein expression level in rats and to evaluate the prognosis of geriatric depression changes associated with hippocampal angiogenesis and intracellular signal transduction molecules Rafael Xin.
5. using multiple linear regression analysis, MVD and evaluation of depression behavior score index of horizontal movement of the open field test, vertical motion score, cleaning time and sucrose consumption test of sucrose consumption relation. Bivariate correlation analysis was used to evaluate VEGF protein, ANG1 protein, bFGF protein, the relationship between MVD and VEGF and evaluation; MVD protein and ANG1 protein, the relationship between MVD and bFGF protein. Using linear regression analysis, evaluation of MVD and VEGF protein, the relationship between MVD and bFGF protein.
[results]
1. open field test and sucrose consumption test showed that the model group, Vin Rafa Sin group and saline group and depression model were successfully established. Open field test results: group comparison: model group, Vin Rafa Sin group, saline group in 3W after stress, stress after 5W respectively with the same group before the stress of 1D, 3W and model group the saline group 5W after stress with the same group after stress, horizontal motion score, vertical movement scores were lower (P < 0.05), shorten the cleaning time (P0.05). Comparison between groups: 5W after stress, compared with the control group, the level of sports scores of the Vin Rafa Sin group, vertical motion score (P0.05), reduce the cleaning time shortened (P0.05); model group and saline group level sports scores, vertical motion score decreased significantly (P0.01), cleaning time was significantly shortened (P0.01); compared with the saline group, Vin Rafa Sin group of vertical movement scores increased (P0.05). The sucrose consumption. Results: group comparison: model group and saline group 5W after stress respectively with the same group of 1D before the stress, the same group of 3W after stress, sucrose consumption, reduced sucrose preference (P 0.05). Comparison between groups: 5W after stress, compared with the control group, sucrose model group and saline group. Consumption was significantly reduced (P0.01); compared with the normal saline group, venlafaxine group of sucrose consumption increased (P0.05).
2.HE staining showed that in control group, the hippocampal pyramidal cell layer thick, neat, compact, the number of capillaries in normal saline group; model group and hippocampal pyramidal cell layer thinning, cell gap increased, disorganized, osteoporosis, decrease in the number of capillaries; venlafaxine Zuhai horse pyramidal cell layer is thin, but the cells arranged in neat, close the number of capillaries, increased significantly, the lumen is irregular. Immunohistochemical staining showed that: compared with the control group, model group and saline group hippocampus decreased microvessel density (P0.05); compared with the normal saline group, venlafaxine group hippocampal microvessel density (P0.05).
3. compared with the control group, the expression of VEGF protein and VEGFR2 protein, VEGFmRNA and VEGFR2mRNA in hippocampus of model group and saline group decreased (P0.05): compared with saline group, the expression of VEGF protein and VEGFR2 protein, VEGFmRNA and VEGFR2mRNA increased in hippocampus of venlafaxine group (P0.05).
4. compared with the control group, the expression of ANG1 protein and TIE2 protein, ANG1mRNA and TIE2mRNA increased in hippocampus of model group and saline group (P0.05). Compared with saline group, the expression of ANG1 protein and TIE2 protein, ANG1mRNA and TIE2mRNA increased in hippocampus of venlafaxine group (P0.05).
5. compared with the control group, the expression of bFGF protein and FGFR1 protein, bFGFmRNA and FGFR1mRNA in hippocampus of model group and saline group decreased (P0.05), compared with the saline group, the expression of bFGF protein and FGFR1 protein, bFGFmRNA and FGFR1mRNA increased significantly in the venlafaxine group (P0.01).
6. compared with the control group, model group and saline group the expression of P13K protein and Akt protein, p-Akt protein, PI3KmRNA and AktmRNA decreased (P0.05); compared with the normal saline group, venlafaxine group the expression of P13K protein and Akt protein, p-Akt protein, PI3KmRNA and AktmRNA expression increased significantly (P0.01).
7. there was no significant difference in the expression of JAK1 and STAT3 protein, JAK1mRNA and STAT3mRNA in the four groups (P0.05).
8. the results in MVD and behavior in regression analysis, significant vertical motion score coefficient of open field experiment (P0.05), the regression equation is MVD=5.056+0.580 vertical motion score. ANG1 protein in VEGF protein, and correlation analysis of bFGF protein, VEGF protein and ANG1 protein, VEGF protein and bFGF protein were significantly correlated (r were 0.573,0.742 and P respectively, 0.003,0.000). In the regression analysis of MVD and VEGF protein, VEGF protein had a statistically significant coefficient (P0.01), the regression equation was MVD=3.391+11.507VEGF. in regression analysis of MVD and bFGF protein, bFGF protein had a statistically significant coefficient (P0.01), the regression equation of MVD=3.047+10.884bFGF.
[Conclusion]
1. this experiment successfully established the model of depression in aged rats. Rats with senile depression depressive like symptoms: psychomotor retardation symptoms, and with the stress time prolonged; the declining interest symptoms appear later. The same dose of venlafaxine for different symptoms curative effect: there are differences between the aggravation of symptoms can curb the activity of hysteresis, but can not be restored to normal levels; prevention and treatment of declining interest symptoms.
In 2. elderly chronic stress depression rats decreased microvessel density and angiogenesis may be inhibited. Venlafaxine intervention of hippocampal microvascular density increased, may contribute to angiogenesis. Hippocampus hippocampus of aged rats with depression and microvessel density in aged rats with depression like symptoms, suggesting that elderly depressive disorder may be related to the damage of blood vessels.
3. after 5 weeks of improved chronic unpredictable mild stress with solitary, VEGF and VEGFR2 receptor in hippocampus in aged depression rats, ANG1 receptor and TIE2 protein, and mRNA and bFGF receptor FGFR1 expression can be changed. 14 days of antidepressant venlafaxine Scripture can promote VEGF and VEGFR2 receptor in hippocampus of elderly depression in rats, ANG1 receptor and TIE2 protein and mRNA expression of bFGF and receptor FGFR1. The expression of VEGF protein and ANG1 protein, VEGF protein and bFGF protein in hippocampus. Microvessel density and VEGF protein, bFGF protein and microvessel density in hippocampus. In VEGF, ANG1, may be involved in depression of old molecules the mechanism of bFGF in angiogenesis in the pathogenesis of venlafaxine and antidepressant effect.
The 4. signal is correlated with angiogenesis in intracellular pathway, pathway of chronic stress depression rats hippocampal PI3K/Akt signal may be affected, venlafaxine can promote the activation of PI3K/Akt signaling pathway.JAK1/STAT3 signaling pathway did not change significantly. The incidence of venlafaxine and may be involved in the elderly depression suggest that PI3K/Akt signal pathway in vascular the new molecular mechanism of antidepressant effect.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.4

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相關(guān)期刊論文 前8條

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